Microarray Technology

Microarray Outreach Program

Vermont Genetics Network
University of Vermont
Outline of the lecture
• Overview of Microarray Technology
• Types of Microarrays
• Manufacturing
• Instrumentation and Software
• Data Analysis-Basic
• Applications
 Microarray Development
• Relatively young technology
Widely adopted

Mainly used in gene discovery

 Evolution & Industrialization
1989: First Affymetrix Genechip Prototype

1994: First Commercial Affymetrix Genechip

1994- First cDNAs arrays were developed at Stanford University.

1994: First Commercial Scanner-Affymetrix

1996- Commercialization of arrays

1997-Genome-wide Expression Monitoring in S. cerevisiae

 What are Microarrays?
• Microarrays are simply small glass or silicon slides upon
the surface of which are arrayed thousands of features
(usually between 500 up to a million)

• Using a conventional hybridization process, the level of

expression of genes is measured (for instance)

• Microarrays are read using laser-based fluorescence

scanners

• The process is “high throughput”

 Why use Microarrays?
• Determine what genes are active in a cell and at what levels

• Compare the gene expression profiles of a control vs treated

• Determine what genes have increased or decreased in during an

experimental condition

• Determine which genes have biological significance in a system

• Discovery of new genes, pathways, and cellular trafficking

 Why analyze so many genes?
• Just because we sequenced a genome doesn’t mean
we know anything about the genes. Thousands of
genes remain without an assigned function.

• Patterns or clusters of genes are more informative

regarding total cellular function than looking at one
or two genes – can figure out new pathways
The six steps in development of a DNA
microarray experiment
1. Manufacturing of the microarray

2. Experimental design and choice of reference: what to

compare to what?

3. Target (sample) preparation and hybridization

4. Image acquisition (scanning) and quantification of

gene expression
5. Database building, filtering, and normalization

6. Bioinformatics: Statistical analysis, data mining, pathway

analysis
 Types of Microarrays

-Expression Arrays
-Protein microarrays (Proteomics)
-Resequencing arrays
-CGH arrays- Comparative genomic hybridization
-SNP Arrays
-Antibody Arrays
-Exon arrays-Alternative splice variant detection
-Tissue Arrays
Microarray Formats

A) Cartridge-based
Spotted
Electronic

B) Spotted Glass Slide

C) Tissue Section Slide
 Cartridge-based Chips

-Miniaturized, high density arrays of DNA oligos

within a plastic housing

-One sample=One chip

(Affymetrix, Agilent, Applied Biosystems…)

- Generally used with expression and DNA arrays

 Cartridge-based Expression Microarrays

Involves Fluorescently tagged biotinylated

cRNA

-One chip per sample

-Uses single fluorescent dye
-More expensive

Affymetrix GeneChip Image

 Spotted Glass Arrays
Uses cDNA, Oligonucleotide, protein, antibody
-Robotically spotted cDNAs or Oligonucleotides
- Printed on Nylon, Plastic, or Glass microscope slide

BD’s Antibody Array

Agilent:Oligonucleotide Array
 Spotted cDNA and Oligo Glass Arrays:
Involves two dyes on the same slide
• Red dye-Cy5
• Green dye-Cy3
• Control and experimental cDNA on same chip
Electroniconically Addressable Microarrays

Nanogen- Nanochip Motorola- eSensor Chip

 Expression Arrays
Most common type of microarray

Spotted glass, cartridge, and electronic

Involves extracting RNA from a sample and converting it to

cDNA by priming off of the Poly A tail of mRNA for eukaryotes
and using random hexamers for prokaryotes [WHY?]

Measures the amount and type of mRNA transcripts

Provides information on whether genes are up or down regulated

in a specific condition
 Protein Microarrays
True protein microarrays are evolving very slow and only a few exist.

Technology is not straight forward due to inherent characteristic of

proteins [e.g. available ligands, folding, drying…]

Most are designed to detect antibodies or enzymes in a biological

system

Protein is on the microarray

Some detect protein-protein interaction by surface plasmon resonance

other use a fluorescence based approach
 Protein Microarrays
The Invitrogen Human Protein Microarray is a high-density microarray

It contains thousands of unique human proteins

[kinases, phophatases, GPCRs, nuclear receptors, and proteases]

 Antibody Arrays
-Assay hundreds of native proteins simultaneously
-Compare protein abundances in a variety of
biological samples
-GenTel and BD biosciences
-Antibody or ligand is on the microarray
Antibody Arrays-labeling scheme
 SNP, Genotyping, and DNA Mapping Arrays
Targets DNA not RNA like expression

Requires amplification of target DNA

Uses multiple probes sets to determine base change at a specific nucleotide

position in the genomic DNA.

Use thousand of oligos that “tile” or span the genomic DNA for
characterization.

Provides sequence and genotyping data including LOH, Linkage analysis and
single nucleotide polymorphisms
 Resequencing Arrays [Affy]

Enable the analysis of up to 300,000+ bases of double-stranded

sequence (600,000 bases total) on a single Affy array

Used for large-scale resequencing of organisms genome and

organelles

Faster and cheaper than sequencing but very limited to few

organisms and/or organelles

Large potential
 Exon Arrays-Alternative splice variant detection

Probes are designed for hybridizing to individual exons of genomic DNA

Tissue or development specific splicing leads to normal or expected protein diversity

Defective splicing can lead to disease

 CGH Arrays- Comparative Genomic Hybridization

Provides DNA and chromosomal information

DNA Copy number and allele-specific information

Determine regions of chromosomal deletion (LOH) or

amplification

Enables the identification of critical gene(s) that have altered

copy number and may be responsible for the development and
progression of a particular disease.
 Tissue Arrays

Slide based “spotted” tissues (not really)

 Assembling Tissue Arrays
Coring of embedded paraffin tissues and plugging or inserting into
new paraffin block

Sectioning and deposition onto a slide

 GeneChip Technology
Affymetrix Inc

Miniaturized, high density arrays of 1,300,000 DNA oligos 1-cm

by 1-cm

Manufacturing Process:

Solid-phase chemical synthesis and Photolithographic

fabrication techniques employed in semiconductor industry

WE WILL DISCUSS THIS IN DETAIL IN ANOTHER PPT

Printed cDNA or Oligonucleotide Arrays
Robotically spotted cDNAs (50mer) or Oligonucleotides
(70mers) vs. Affymetrix’s that uses 25mers

• Printed on Nylon, Plastic, or Glass surface

cDNA Array

Microarray of
thousands of Oligos
on a glass slide
steel
Spotted arrays
spotting pin

chemically modified slides

384 well source

plate
1 nanolitre spots
90-120 um diameter
 The process
Building the chip:
MASSIVE PCR PCR PURIFICATION
and PREPARATION

PREPARING SLIDES PRINTING

RNA
preparation: Hybing the
CELL CULTURE
AND HARVEST chip: POST PROCESSING

ARRAY HYBRIDIZATION
RNA ISOLATION

DATA ANALYSIS

cDNA PRODUCTION PROBE LABELING

 Building the chip

PCR amplification Consolidate into

Arrayed Library
(96 or 384-well plates of
bacterial glycerol stocks)
Directly from colonies with 384-well plates
SP6-T7 primers in 96-well
plates

Spot as microarray
on glass slides
Hybridization chamber
3XSSC

HYB CHAMBER

ARRAY

LIFTERSLIP

SLIDE
LABEL
SLIDE LABEL

• Humidity
• Temperature
• Formamide
(Lowers the Tm)
 Expression profiling with cDNA microarrays
cDNA “A” cDNA “B”
Cy5 labeled Cy3 labeled

Laser 1 Laser 2

Hybridization Scanning

Analysis Image Capture

Image analysis of cDNA array
 Spotted cDNA microarrays
Advantages
-Lower price and flexibility

-Simultaneous comparison of two related biological samples

(tumor versus normal, treated versus untreated cells)

Disadvantages
-Needs sequence verification
-Measures the relative level of expression between 2 samples
-Features can come off the surface-poor adhesion
-Labor Intensive, requires designated staff and equipment
- Data is can be variable
 Microarray data analysis
Scatter plots, significance analysis, clustering,
pathway analysis… to name a few

• Intensities of experimental samples versus

normal samples

• Quick look at the changes and overall quality of

microarray
Normal vs. Normal Normal vs. Tumor
Bioinformatics : Microarray data analysis
- Often is a stand alone dept. within an institute (such as
the case at UVM), but works very closely with a
microarray facility

- A whole field by itself

- Involves extensive knowledge of gene ontology,

biochemical pathways, gene annotation, cell signaling,
cellular trafficking , ….

- Uses special software such as Spotfire, Genesifter,

Genespring…to name a few.
 Validating Microarray Expression Data

Microarray data are not stand alone results and requires

validation by second method

Microarray data is only semi-quantitative because of a limited

dynamic range.

True quantitative results must be determined with another

technique such as Quantitative real-time PCR
 Microarray Validation
Two types of validation
1] Validating the instrument data using the same RNA (confirming a
result)
And most importantly
2] Validating the biological phenomenon with new samples same
experiment conditions
Methods
Northern Blots, RPA’s, Immunohistochemistry,Western Blot, in silico
PCR- i.e.Quantitative real-time PCR

**DNA mapping Arrays or CGH may also help indicate where or why a change is
occuring
 Microarray Applications

• Identify new genes implicated in disease progression and

treatment response (90% of our genes have yet to be ascribed a
function)

• Assess side-effects or drug reaction profiles

• Extract prognostic information, e.g. classify tumors based on

hundreds of parameters rather than 2 or 3.

• Identify new drug targets and accelerate drug discovery and

testing
 Microarray Applications

• Gene Discovery-
– Assigning function to sequence
– Discovery of disease genes and drug targets
– Target validation
• Genotyping
– Patient stratification (pharmacogenomics)
– Adverse drug effects (ADE)
• Microbial ID
 Microarray Future
Diagnostics -[Affy, Nanogen only at this time]
– Disease detection
– Tumor classification
– Patient stratification
– Intervention therapeutics

Treatment and Customized Medicine

Clinical arrays currently available are the AmpliChip CYP450 by

Affymetrix and Roche. Used for predictive phenotyping in defects of the
cytochrome P450 Genes
 Conclusion
• Technology is evolving rapidly

• Blending of biology, automation, and informatics

• New applications are being pursued

– Beyond gene discovery into screening, validation,
clinical genotyping, etc

• Microarrays are becoming more broadly available and

accepted
– Protein Arrays, tissue arrays, etc
– Diagnostic Applications
 W.W.W. resources
• Complete guide to “microarraying”
http://cmgm.stanford.edu/pbrown/mguide/

• http://www.microarrays.org
– Parts and assembly instructions for printer and scanner;
– Protocols for sample prep;
– Software;
– Forum, etc.

• Animation: http://www.bio.davidson.edu/courses/genomics/chip/chip.html